Stimulation of synaptic N-methyl-D-aspartate-type glutamate receptors (NMDARs) triggers neuronal survival- promoting signaling pathways, whereas activation of extrasynaptic NMDARs initiates death-promoting pathways. We recently showed that an imbalance between synaptic and extrasynaptic NMDAR activity promotes the pathogenesis of Huntington's disease (Okamoto et al. Nat. Med., 2009). Notably, a perturbed balance in synaptic vs. extrasynaptic NMDAR activity has been found in other neurological diseases, including Alzheimer's disease (AD). The pathogenic downstream signaling pathways activated by such an imbalance are potential targets for therapeutic intervention in neurodegenerative diseases. Nonetheless, the signaling events downstream of synaptic vs. extrasynaptic NMDARs remain incompletely characterized. To explore these signaling networks, we propose to develop improved multidimensional liquid chromatography- (MDLC) tandem mass spectrometry- (MS/MS) based (phospho)proteomic (combined proteomic and phosphoproteomic) technologies since protein phosphorylation is key to cell signaling. Dynamic and reversible site-specific phosphorylation controls myriad signaling events. Advances in MDLC methods, including phosphopeptide enrichment, plus faster, more sensitive and accurate mass spectrometers, with several peptide fragmentation modes, make it increasingly feasible to quantify even low abundance (phospho)proteins, as we have found in our preliminary work. However, comprehensive (phospho)proteomic datasets are not yet attainable. Thus, we propose new, complementary approaches in (phospho)proteomic technologies to elucidate the phosphorylation networks activated by synaptic vs. extrasynaptic NMDARs. This application includes two specific aims: 1) To obtain unparalleled depth of (phospho)proteome coverage to elucidate the control of synaptic vs. extrasynaptic signaling in primary cerebrocortical neurons by development and application of more comprehensive MDLC-MS/MS; and, 2) To apply high throughput, quantitatively precise, targeted (phospho)proteomic tools to analyze phosphoprotein changes in synaptic vs. extrasynaptic signaling in the brains of mouse models of neurodegenerative diseases such as AD using targeted mass spectrometry. In Aims 1 and 2, we will develop the tools to quantify the phosphoproteome of synaptic vs. extrasynaptic NMDAR downstream signaling with the goal of comprehensive coverage of the networks in diseased tissues. Key phosphorylation networks will be experimentally verified using molecular/cellular biology techniques, and the data provided to the community as a resource for further validation and biological application. Also, we will identify differences in synaptic vs. extrasynaptic NMDAR-triggered phosphorylation networks in mouse models of neurodegenerative diseases by high throughput approaches (Aim 2). Importantly, these data will enable us to quantitatively elucidate abnormal or imbalanced phosphorylation networks in order to identify novel targets for therapeutic intervention in neurodegenerative diseases.